Genome-wide characterization, expression analyses, and functional prediction of the NPF family in Brassica napus.

BACKGROUND: NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER (NRT1/PTR) family (NPF) members are essential transporters for many substrates in plants, including nitrate, hormones, peptides, and secondary metabolites. Here, we report the global characterization of NPF in the important oil crop Brassica napus, including that for phylogeny, gene/protein structures, duplications, and expression patterns. RESULTS: A total of 199 B. napus (BnaNPFs) NPF-coding genes were identified. Phylogenetic analyses categorized these genes into 11 subfamilies, including three new ones. Sequence feature analysis revealed that members of each subfamily contain conserved gene and protein structures. Many hormone-/abiotic stress-responsive cis-acting elements and transcription factor binding sites were identified in BnaNPF promoter regions. Chromosome distribution analysis indicated that BnaNPFs within a subfamily tend to cluster on one chromosome. Syntenic relationship analysis showed that allotetraploid creation by its ancestors (Brassica rapa and Brassica oleracea) (57.89%) and small-scale duplication events (39.85%) contributed to rapid BnaNPF expansion in B. napus. A genome-wide spatiotemporal expression survey showed that NPF genes of each Arabidopsis and B. napus subfamily have preferential expression patterns across developmental stages, most of them are expressed in a few organs. RNA-seq analysis showed that many BnaNPFs (32.66%) have wide exogenous hormone-inductive profiles, suggesting important hormone-mediated patterns in diverse bioprocesses. Homologs in a clade or branch within a given subfamily have conserved organ/spatiotemporal and hormone-inductive profiles, indicating functional conservation during evolution. qRT-PCR-based comparative expression analysis of the 12 BnaNPFs in the NPF2-1 subfamily between high- and low-glucosinolate (GLS) content B. napus varieties revealed that homologs of AtNPF2.9 (BnaNPF2.12, BnaNPF2.13, and BnaNPF2.14), AtNPF2.10 (BnaNPF2.19 and BnaNPF2.20), and AtNPF2.11 (BnaNPF2.26 and BnaNPF2.28) might be involved in GLS transport. qRT-PCR further confirmed the hormone-responsive expression profiles of these putative GLS transporter genes. CONCLUSION: We identified 199 B. napus BnaNPFs; these were divided into 11 subfamilies. Allopolyploidy and small-scale duplication events contributed to the immense expansion of BnaNPFs in B. napus. The BnaNPFs had preferential expression patterns in different tissues/organs and wide hormone-induced expression profiles. Four BnaNPFs in the NPF2-1 subfamily may be involved in GLS transport. Our results provide an abundant gene resource for further functional analysis of BnaNPFs.

Genome-Wide Survey and Expression Analysis of the KT/HAK/KUP Family in Brassica napus and Its Potential Roles in the Response to K+ Deficiency.

The KT/HAK/KUP (HAK) family is the largest potassium (K+) transporter family in plants, which plays key roles in K+ uptake and homeostasis, stress resistance, and root and embryo development. However, the HAK family has not yet been characterized in Brassica napus. In this study, 40 putative B. napus HAK genes (BnaHAKs) are identified and divided into four groups (Groups I-III and V) on the basis of phylogenetic analysis. Gene structure analysis revealed 10 conserved intron insertion sites across different groups. Collinearity analysis demonstrated that both allopolyploidization and small-scale duplication events contributed to the large expansion of BnaHAKs. Transcription factor (TF)-binding network construction, cis-element analysis, and microRNA prediction revealed that the expression of BnaHAKs is regulated by multiple factors. Analysis of RNA-sequencing data further revealed extensive expression profiles of the BnaHAKs in groups II, III, and V, with limited expression in group I. Compared with group I, most of the BnaHAKs in groups II, III, and V were more upregulated by hormone induction based on RNA-sequencing data. Reverse transcription-quantitative polymerase reaction analysis revealed that the expression of eight BnaHAKs of groups I and V was markedly upregulated under K+-deficiency treatment. Collectively, our results provide valuable information and key candidate genes for further functional studies of BnaHAKs.

Adsorption of Cu2+ and mechanism by natural biofilm.

The biofilm culturing device fixed on the slides was vertically placed in the commonly called small Li Lake of Jiangnan University. The adsorption experiment of Cu2+ was carried out by mature biofilm. Besides, scanning electron microscope (SEM), polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy-energy spectrum (SEM-EDX) were used to analysis the effect of Cu2+ on the morphological structure of biofilm. The result indicated that when the initial concentration of Cu2+ was 5 mg·L-1, the absorption capacity of Cu2+ by unit mass biofilm is the maximum. More extracellular polymeric substances (EPS) were released by biofilm due to the stimulation of Cu2+. EPS was beneficial to the adsorption of Cu2+ by biofilm. After the adsorption of Cu2+, the bacterial diversity index decreased, while there were no significant differences in microbial communities on biofilm. Moreover, the main groups combining Cu2+ were the hydroxyl groups and amide groups in S-EPS and B-EPS. Ion exchange is a mechanism of the adsorption of Cu2+ by EPS.

Optimization of Demineralization and Pyrolysis Performance of Eucalyptus Hydrothermal Pretreatment.

The preparation of bio-oil through biomass pyrolysis is promoted by different demineralization processes to remove alkali and alkaline earth metal elements (AAEMs). In this study, the hydrothermal pretreatment demineralization was optimized by the response surface method. The pretreatment temperature, time and pH were the response elements, and the total dissolution rates of potassium, calcium and magnesium were the response values. The interactions of response factors for AAEMs removal were analyzed. The interaction between temperature and time was significant. The optimal AAEMs removal process was obtained with a reaction temperature of 172.98 °C, time of 59.77 min, and pH of 3.01. The optimal dissolution rate of AAEMs was 47.59%. The thermal stability of eucalyptus with and without pretreatment was analyzed by TGA. The hydrothermal pretreatment samples exhibit higher thermostability. The composition and distribution of pyrolysis products of different samples were analyzed by Py-GC/MS. The results showed that the content of sugars and high-quality bio-oil (C6, C7, C8 and C9) were 60.74% and 80.99%, respectively, by hydrothermal pretreatment. These results show that the removal of AAEMs through hydrothermal pretreatment not only improves the yield of bio-oil, but also improves the quality of bio-oil and promotes an upgrade in the quality of bio-oil.

Superior separation of hemicellulose-derived sugars from eucalyptus with tropic acid pretreatment.

Organic acid pretreatments can efficiently separate biomass-based hemicellulose and selectively produce hemicellulose-derived sugars. In this study, hemicellulose is separation as xylose, oligosaccharides in the tropic acid-catalyzed hydrothermal pretreatment of eucalyptus. The maximum yield of hemicellulose-derived sugars (85.78 %) with 71.25 % xylose selectivity (based on the total xylose in raw material) was achieved in the hydrolysate under optimal conditions (5 % TA, 160 ℃, 80 min). The yield of hemicellulose-derived sugar and the separation yield of hemicellulose increased by 11.06 % and 11.45 % compared with glycolic acid pretreatment in the similar severity factor. The separation yield of cellulose and lignin was decreased by 4.23 % and 0.98 %, respectively. This resulted in residual solids with higher biological stability (higher fiber crystallinity index, higher thermal stability, and higher lignin content). Therefore, higher hemicellulose separation selectivity and rich hemicellulose-derived sugars were obtained using TA pretreatment. The work would bring up a new method for biomass refining.

Enhancement of separation selectivity of hemicellulose from bamboo using freeze-thaw-assisted p-toluenesulfonic acid treatment at low acid concentration and high temperature.

The cellulose-rich residual solids are obtained with p-toluenesulfonic acid (p-TsOH) treatment. However, better fractionation of hemicellulose and separation is difficult to obtain during treatment. This study aims at investigating the separation selectivity of bamboo hemicellulose using freeze-thaw-assisted p-TsOH (F/p-TsOH) treatment. The desired separation effect was achieved at freezing temperature -40 °C, freezing time 20 h, p-TsOH concentration 3.0 %, treatment temperature 130 °C and time 80 min. 93.26 % hemicellulose separation was found, which was 32.88 % higher than that of conventional p-TsOH treatment. Furthermore, the separation yield of lignin decreased significantly from 69.29 % to 13.98 %. The distinct lignin characteristic absorption peaks were found, while that of hemicellulose was difficult to observe. The fiber crystallinity index increased from 50.42 to 56.55 %. Furthermore, greater selectivity for hemicellulose separation was achieved. The results provide a new research thinking for efficient fractionation of lignocellulosic biomass by organic acid treatment.

Efficient removal of residual lignin from eucalyptus pulp via high-concentration chlorine dioxide treatment and its effect on the properties of residual solids.

In fact, effectively removing lignin from pulp fibers facilitates the conversion and utilization of cellulose. In this study, the residual lignin in eucalyptus pulp was separated using a high concentration of chlorine dioxide. The effects of chlorine dioxide dosage, temperature, and time on lignin removal were investigated. The optimal conditions are chlorine dioxide dosage 5.0%, reaction temperature 40 °C, and reaction time 30 min. The lignin removal yield is 88.21%. The removal yields of cellulose and hemicellulose are 2.28 and 17.00%, respectively. The treated eucalyptus pulp has higher fiber crystallinity and thermal stability. The carbon content on the fiber surface is significantly reduced. The results show that lignin is removed by efficient oxidation, and the degradation of carbohydrates is inhibited using high concentrations of chlorine dioxide at low temperatures and short reaction times. This provides theoretical support for high value conversion of cellulose.

Effect of temperature on simultaneous separation and extraction of hemicellulose using p-toluenesulfonic acid treatment at atmospheric pressure.

Hemicelluloses were effectively separated using p-toluenesulfonic acid (p-TsOH) treatment at high temperature. High temperature and pressure promoted hydrolysis of hemicellulose, which limited its value upon recovery. In this study, bagasse hemicellulose was separated and extracted by p-TsOH treatment at atmospheric pressure. The effects of temperature, p-TsOH concentration, and time on hemicellulose separation and extraction were investigated. The optimal conditions were 80 °C, 3.0% p-TsOH, and 120 min. The separation and extraction yield of hemicellulose was 73.23% and 36.02%, respectively. Extraction hemicellulose with 95.60% purity was obtained. In addition, the dissolution mechanism of hemicellulose was analyzed. Degradation of ß-glycosidic bonds was inhibited. Benzyl ether bond between carbohydrates and lignin was selectively cleaved. The skeleton structure of xylan in hemicellulose was protected while the functional groups of branch chain were severely damaged. It provides a valuable theoretical basis for the efficient separation and extraction of hemicellulose.

Application and prospect of organic acid pretreatment in lignocellulosic biomass separation: A review.

As a clean and efficient method of lignocellulosic biomass separation, organic acid pretreatment has attracted extensive research. Hemicellulose or lignin is selectively isolated and the cellulose structure is preserved. Effective fractionation of lignocellulosic biomass is achieved. The separation characteristics of hemicellulose or lignin by different organic acids were summarized. The organic acids of hemicellulose were separated into hydrogen ionized, autocatalytic and α-hydroxy acids according to the separation mechanism. The separation of lignin depends on the dissolution mechanism and spatial effect of organic acids. In addition, the challenges and prospects of organic acid pretreatment were analyzed. The separation of hemicellulose and enzymatic hydrolysis of cellulose were significantly affected by the polycondensation of lignin, which is effectively inhibited by the addition of green additives such as ketones or alcohols. Lignin separation was improved by developing a deep eutectic solvent treatment based on organic acid pretreatment. This work provides support for efficient cleaning of carbohydrate polymers and lignin to promote global carbon neutrality.

High efficiency and clean separation of eucalyptus components by glycolic acid pretreatment.

Glycolic acid has chemical properties similar to those of formic acid. Therefore, similar to formic acid pretreatment, glycolic acid pretreatment has the separation effect of hemicellulose. In this study, eucalyptus hemicellulose was effectively separated by glycolic acid pretreatment. The effects of glycolic acid concentration, temperature and time on the separation of cellulose, hemicellulose and lignin were investigated. The optimum conditions were acid concentration 5.40%, temperature 140 °C, time 3.0 h. The highest yield of xylose was 56.72%. The recovery rate of glycolic acid was 91%. Compared to formic acid, the yield of xylose increased to 10.33% while that of lignin decreased to 11.08%. It showed high selectivity for hemicellulose separation, yielding 65.48% hemicellulose with 72.08% purity. The depolymerization and repolymerization of lignin were inhibited. The integrity of the cellulose structure was preserved. It provides theoretical support for the fractional separation and high-value transformation of lignocellulosic biomass.

Effects of oxygen tension on the microbial community and functional gene expression of aerobic methane oxidation coupled to denitrification systems.

Aerobic CH4 oxidation coupled to denitrification (AME-D) can not only mitigate the emission of greenhouse gas (e.g., CH4) to the atmosphere, but also reduce NO3- and/or NO2- and alleviate nitrogen pollution. The effects of O2 tension on the community and functional gene expression of methanotrophs and denitrifiers were investigated in this study. Although higher CH4 oxidation occurred in the AME-D system with an initial O2 concentration of 21% (i.e., the O2-sufficient condition), more NO3--N was removed at the initial O2 concentration of 10% (i.e., the O2-limited environment). Type I methanotrophs, including Methylocaldum, Methylobacter, Methylococcus, Methylomonas, and Methylomicrobium, and type II methanotrophs, including Methylocystis and Methylosinus, dominated in the AME-D systems. Compared with type II methanotrophs, type I methanotrophs were more abundant in the AME-D systems. Proteobacteria and Actinobacteria were the main denitrifiers in the AME-D systems, and their compositions varied with the O2 tension. Quantitative PCR of the pmoA, nirS, and 16S rRNA genes showed that methanotrophs and denitrifiers were the main microorganisms in the AME-D systems, accounting for 46.4% and 24.1% in the O2-limited environment, respectively. However, the relative transcripts of the functional genes including pmoA, mmoX, nirK, nirS, and norZ were all less than 1%, especially the functional genes involved in denitrification under the O2-sufficient condition, likely due to the majority of the denitrifiers being dormant or even nonviable. These findings indicated that an optimal O2 concentration should be used to optimize the activity and functional gene expression of aerobic methanotrophs and denitrifiers in AME-D systems.